Activity analysis method of tea tree F3 'H promoter

Abstract

The invention belongs to but is not limited to the technical field of gene analysis, and particularly relates to an activity analysis method of a tea tree F3 'H promoter, which comprises the following steps: S1, cloning F3' HP1 and a 5'end fragment deletion promoter sequence thereof, and constructing an expression vector; s2, analyzing the cis-acting element of the F3 'HP1 and the 5'end fragment of the F3' HP1 without the promoter sequence; s3, carrying out GUS histochemical staining and GUS enzyme activity determination on the instantly converted tobacco; and S4, response of the F3 'H to low-temperature treatment. The result shows that the promoter sequence (F3' HP1) of the upstream 1806bp of the F3 'H gene' ATG 'is obtained through cloning, and four 5' fragment-deleted F3 'H gene promoter sequences, namely F3' HP2, F3 'HP3, F3' HP4 and F3 'HP5 are obtained, and the sequence lengths are 804, 479bp, 195 and 154bp respectively, the F3' HP1 sequence also has response elements related to low temperature, light, gibberellin and the like besides basic elements of TATA-box and CAAT-box, and the transcriptome data result shows that the F3 'H responds to low temperature.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of gene analysis technology, and in particular relates to a tea plant F3'H promoter, a cloning method and an application thereof. Background Art

[0002] Tea trees are evergreen leaf crops, and tea made from their new shoots is one of the three major non-alcoholic beverages in the world (Wan Xiaochun, 2003; Zhou et al., 2019). Tea polyphenols usually account for 18%-36% of the dry weight of tea leaves, of which catechins are the main components of tea polyphenol compounds, accounting for 70%-80% of tea polyphenols. They have physiological functions such as antioxidant, anti-inflammatory, improvement of cardiovascular and cerebrovascular disease symptoms, prevention of diabetes, and protection of the nervous system (Wan Xiaochun, 2003; Qu et al., 2021). According to the number of hydroxyl groups on the B ring, catechins can be divided into trihydroxycatechins (epigallocatechin gallate, epigallocatechin, epigallocatechin gallate; EGCG, EGC and GC) and dihydroxycatechins (epicatechin gallate, epicatechin and epigallocatechin; ECG, EC and C) (Xia et al., 2009; Jin et al., 2014), among which EGCG, ECG, EGC and EC are the most abundant, accounting for more than 95% of the total catechins in tea (Jin et al., 2014). Dihydroxycatechins and trihydroxycatechins are the material basis for the formation of theaflavins, the quality components of black tea. A higher catechin index (CI) [(EC+ECG) / (EGC+EGCG)] can increase the ratio of theaflavins / thearubigins in black tea, thereby improving the quality of black tea (Robertson, 1983; Kottur et al., 2010; Xu et al., 2018; Jin et al., 2018).

[0003] Jin et al. (2014) showed that the CI values ​​of tea resources in my country are generally low (Jin et al., 2014; Liu Yufei et al., 2021), mainly because the dihydroxylated catechin content is low. Therefore, increasing the content of dihydroxylated catechins has become an important research direction for the current breeding of black tea varieties. The synthesis of catechins in tea trees requires a series of enzymatic oxidation reactions, among which flavonoid 3'-hydroxylase (F3'H) is a key enzyme in the catechin synthesis pathway, catalyzing the hydroxylation of flavonoids at the 3' position to generate dihydroxylated catechin components ECG, EC and C (Pang et al., 2021). In-depth research on the F3'H gene from the aspects of protein function, protein modification, transcriptional regulation and post-transcriptional regulation can provide theoretical support for improving the content of dihydroxylated catechins in tea trees.

[0004] Studies have shown that the catalytic substrates of F3'H enzyme in tea plants are diverse, and it can catalyze the conversion of naringenin, kaempferol and dihydrokaempferol into eriodictyol, quercetin and dihydroquercetin, respectively (Guo Lina, 2019). In addition, the expression of F3'H gene in tea plants is affected by hormones (Shi et al., 2015), light (Zheng et al., 2019; Liu et al., 2018; Wang et al., 2022; Zhao et al., 2023), temperature (Shen et al., 2015) and other factors. Although the function of F3'H in tea plants has been studied in depth, and its response to external factors has also been analyzed, there have been no reports on its promoter activity and the key active segments of the promoter. The promoter is the core of controlling gene expression and is the sequence that transcription factors specifically bind to. The study of its activity is conducive to exploring the transcriptional regulation mechanism of genes (Vandel et al., 2019; Martinez-Corral et al., 2023). Therefore, the present invention cloned the promoter of the F3'H gene in tea plants, deleted the 5' end fragment of its full-length sequence, and then studied the promoter activity of F3'H through bioinformatics analysis, transient transformation of tobacco, GUS histochemical staining and GUS protein quantification, laying a foundation for exploring the key active sequence of the F3'H gene and further revealing the transcriptional regulation mechanism of the F3'H gene. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for analyzing the activity of tea plant F3'H promoter.

[0006] The present invention is achieved by a method for analyzing the activity of tea plant F3'H promoter, comprising:

[0007] Cloning and expression vector construction of S1, F3'HP1 and its 5' end fragment lacking promoter sequence;

[0008] S2, cis -acting element analysis of the promoter sequence of F3'HP1 and its 5' end fragment deletion;

[0009] S3, GUS histochemical staining and GUS enzyme activity assay of transiently transformed tobacco;

[0010] S4, Response of F3'H to low temperature treatment.

[0011] Further, S1 specifically includes: referring to the upstream sequence of the start codon of the F3'H gene in the genome, designing specific primers, cloning a 1806bp promoter sequence from the sample 'Yunkang No. 10' (YK10) by PCR, and naming it F3'HP1, the GC content of which is 32%. According to the cloned F3'HP1 sequence, designing 5' fragment deletion primers, and finally obtaining 4 fragment deletion sequences, which are named F3'HP2, F3'HP3, F3'HP4 and F3'HP5 according to their lengths, and the sequence lengths are 804, 479, 195 and 154bp, respectively.

[0012] Further, S2 specifically includes: using the online software PlantCARE, the cis-acting elements of the five promoter sequences were predicted and analyzed. The results showed that the F3'HP1 sequence had multiple TATA-boxes and CAAT-boxes, and had multiple other types of cis-acting elements, such as light response elements (Box 4, Sp1, TCCC-motif and TCT-motif), anaerobic induction related elements (ARE and GC-motif), low temperature response element (LTR), and gibberellin response element (P-box). With the deletion of the 5' end of the F3'H promoter, the types and number of cis-acting elements on the sequence gradually decreased. For example, starting from F3'HP2, the low temperature response element (LTR) and the anaerobic induction related elements (ARE and GC-motif) were both missing; F3'HP4 and F3'HP5 only had TATA-box, and F3'HP5 had even fewer TATA-boxes. The gradual decrease in the types and number of cis-acting elements of the F3'H promoter may affect the activity of the F3'H promoter.

[0013] Furthermore, S3 specifically includes: performing GUS histochemical staining on the tobacco leaves after transient transformation, the staining of the F3'HP1 and F3'HP2 samples is darker, and F3'HP2 is darker than F3'HP3; the tobacco leaves transformed with F3'HP3 also have obvious blue spots, but the color is significantly weaker than that of the samples transformed with F3'HP1 and F3'HP2; no blue spots were observed in the samples transformed with F3'HP4 and F3'HP5 and the control samples.

[0014] Further, S4 specifically includes: the promoter of the F3'H gene has a low temperature response element (LTR), a gibberellin response element (P-box) and multiple light response elements. When the tea tree is treated at 10℃ for 6h (CA_6h), the F3'H gene is down-regulated; when the tea tree is cultured at 10℃ during the day and 4℃ at night for 7 days (CA_7d), the F3'H gene is significantly up-regulated; when the tea tree is cultured at 4℃ during the day and 0℃ at night for 7 days (FA_7d), the F3'H gene is down-regulated, but the expression level is significantly higher than before cold acclimation (CK, 25℃ during the day, 20℃ at night, 12h photoperiod, relative humidity of 70%); after cold acclimation, the normal CK conditions are restored for 7 days, and the expression level of the F3'H gene is significantly higher than that of CK.

[0015] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0016] First, compared with the full-length promoter F3'HP1, the 5' fragment-deleted F3'HP2 has stronger activity ( Figure 3 ), indicating that the segment between F3'HP1 and F3'HP2 may have a binding site for a transcriptional inhibitor. The results of GUS histochemical staining and enzyme activity assays showed that after the segment between F3'HP2 and F3'HP3 was deleted, the F3'H promoter activity dropped sharply, and the enzyme activity of F3'HP2 was 7.2 times that of F3'HP3, and there was no significant difference between the enzyme activity of F3'HP3 and the control, indicating that the segment between F3'HP2 and F3'HP3 was a key segment to ensure that the F3'H promoter had high activity. Although the enzyme activity of F3'HP3 was not significantly different from that of the control, it can be found from the results of staining and enzyme activity assays that F3'HP3 still had certain promoter activity, while F3'HP4 and F3'HP5 had no detectable activity, indicating that the segment between F3'HP3 and F3'HP4 may contain basic elements that meet the F3'H promoter activity. The present invention's exploration of the key segment of F3'H promoter activity provides a certain reference for analyzing the regulatory mechanism of the F3'H gene.

[0017] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: The inventors discovered the tea tree F3'H gene promoter of the present invention through research on tea trees, a leaf-growing economic crop. The full-length and fragments of the promoter have different activities, and its key active segments have been determined, which can be used in various applications of genetic engineering modification of the content of tea flavonoids, thereby increasing the economic value of tea leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1It is a schematic diagram of the analysis of the number of cis-acting elements of the F3'H promoter fragment provided in an embodiment of the present invention;

[0019] Figure 2 It is a schematic diagram of the analysis of the number of cis-acting elements of the F3'H promoter fragment provided in an embodiment of the present invention;

[0020] Figure 3 Schematic diagram of the analysis of promoter activity of different lengths of F3'H gene provided in the embodiment of the present invention; wherein, a: GUS histochemical staining; b: GUS protein activity;

[0021] Figure 4 This is a schematic diagram of the response of the F3'H gene to low temperature acclimation provided by an embodiment of the present invention;

[0022] Figure 5 The present invention provides a method for analyzing the activity of the tea tree F3'H promoter. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] The invention designs specific primers by referring to the sequence information upstream of the start codon of the F3'H gene in the tea plant genome, and clones the F3'HP1 promoter sequence with a length of 1806 bp from the sample using PCR technology. At the same time, in order to analyze the influence of different fragments of the promoter on the activity, a 5' end deletion primer is further designed, and fragments with lengths of 804, 479, 195 and 154 bp are obtained in sequence, and are named F3'HP2, F3'HP3, F3'HP4 and F3'HP5 respectively. These sequences are inserted into an expression vector for subsequent experiments. The cloning of the promoter sequence and the construction of the fragments provide a basic framework for analyzing its function.

[0025] The online tool PlantCARE was used to analyze the cis-acting elements of the complete F3'HP1 sequence and the fragment-deleted F3'HP2, F3'HP3, F3'HP4 and F3'HP5. The results showed that the complete F3'HP1 sequence contained multiple functional elements, such as TATA-box, CAAT-box, light response element, low temperature response element and gibberellin response element. With the gradual loss of the 5' end fragment, the number and types of cis-acting elements decreased significantly. For example, starting from F3'HP2, the low temperature response element and anaerobic induction-related elements were lost, and F3'HP4 and F3'HP5 only retained a small amount of TATA-box. The reduction of cis-acting elements may have a significant effect on the activity of the promoter, which provides a theoretical basis for subsequent activity analysis.

[0026] The expression vectors constructed with the promoter sequences of each fragment were transferred into tobacco leaves through transient transformation technology, and GUS histochemical staining and enzyme activity assay were performed. The staining results showed that the complete F3'HP1 promoter and F3'HP2 samples were stained darker and had higher activity; although the F3'HP3 samples also had blue spots, they were significantly weaker than those of F3'HP1 and F3'HP2; while no obvious blue spots were observed in the F3'HP4 and F3'HP5 samples and the control samples. This indicates that the loss of the 5' end of the promoter leads to a gradual weakening of its activity, and the loss of key cis-acting elements such as TATA-box may be the main reason affecting the activity.

[0027] By designing different low temperature treatment experiments, the response characteristics of the F3'H promoter to low temperature conditions were verified. After being treated at 10℃ for 6 hours, the F3'H gene showed down-regulated expression; after being continuously cultured at 10℃ during the day and 4℃ at night for 7 days, the expression level of the F3'H gene was significantly up-regulated; when the temperature was further lowered to 4℃ during the day and 0℃ at night for 7 days, the expression level of the F3'H gene was down-regulated, but still higher than the level before cold acclimation. Cold acclimation treatment makes the F3'H gene more sensitive to low temperature, and it shows a high expression state after returning to normal conditions.

[0028] Through experimental verification of low temperature treatment and cold acclimation, it was found that the low temperature response element contained in the F3'H promoter has a significant regulatory effect on gene expression after cold acclimation. The recovery level of F3'H gene expression after cold acclimation was significantly higher than that of the control group without cold acclimation, which shows that the F3'H promoter can regulate the dynamic changes of gene expression under low temperature stress conditions, and its low temperature response element plays a key role in regulating gene activity.

[0029] The present invention systematically reveals the activity characteristics of the tea tree F3'H promoter and its response rules to environmental factors through the construction of the complete promoter and its deletion fragments, the analysis of cis-acting elements, the GUS reporter gene activity experiment and the dynamic analysis of low temperature response. The results show that the presence of low temperature response elements in the promoter is the key to regulating gene expression under low temperature stress, and the gradual deletion of the 5' end fragment significantly affects the activity of the promoter. By constructing an activity analysis method, an experimental basis is provided for studying the response mechanism of tea tree to low temperature stress, and a foundation is laid for tea tree breeding and the development of functional genes.

[0030] 1 Results and analysis

[0031] 1.1 Cloning of promoter sequence of F3'HP1 and its 5' end fragment and construction of expression vector

[0032] Specific primers were designed based on the upstream sequence of the start codon of the F3'H gene in the reference genome. A promoter sequence of 1806 bp was cloned from sample YK10 by PCR and named F3'HP1. The GC content of the sequence was 32%. Primers for 5' fragment deletion were designed based on the cloned F3'HP1 sequence. Finally, four sequences after fragment deletion were obtained. They were named F3'HP2, F3'HP3, F3'HP4 and F3'HP5 according to their lengths. The sequence lengths were 804, 479, 195 and 154 bp, respectively ( Figure 1 ).

[0033] Note: The rightmost end is the 3' end of the sequence.

[0034] 1.2 Analysis of cis-acting elements in the promoter sequence of F3'HP1 and its 5' end fragment

[0035] The online software PlantCARE was used to predict and analyze the cis-acting elements of the six promoter sequences. The results showed that the F3'HP1 sequences all had multiple TATA-boxes and CAAT-boxes, as well as multiple other types of cis-acting elements, such as light-responsive elements (Box 4, Sp1, TCCC-motif, and TCT-motif), anaerobic induction-related elements (ARE and GC-motif), low temperature response elements (LTR), gibberellin response elements (P-box), etc. ( Figure 1 , Figure 2 and Table 1). Figure 1 and Figure 2 It was found that with the loss of the 5' end of the F3'H promoter, the types and number of cis-acting elements in the sequence gradually decreased. For example, starting from F3'HP2, both the low temperature response element (LTR) and the anaerobic induction-related elements (ARE and GC-motif) were missing; F3'HP4 and F3'HP5 only had TATA-box, and F3'HP5 had even fewer TATA-boxes. The gradual decrease in the types and number of cis-acting elements of the F3'H promoter may affect the activity of the F3'H promoter.

[0036] Table 1 Functions of different cis-acting elements

[0037]

[0038]

[0039]

[0040] 1.3 GUS histochemical staining and GUS enzyme activity assay of transiently transformed tobacco

[0041] GUS histochemical staining was performed on the tobacco leaves after transient transformation. Figure 3 As shown in a, the staining of F3'HP1 and F3'HP2 samples was darker, and F3'HP2 was darker than F3'HP3; the tobacco leaves transformed with F3'HP3 also had obvious blue spots, but the color was significantly weaker than that of the samples transformed with F3'HP1 and F3'HP2; no blue spots were observed in the samples transformed with F3'HP4 and F3'HP5 and the control samples.

[0042] The results of GUS enzyme activity assay showed that the samples transfected with F3'HP1 and F3'HP2 had strong GUS enzyme activity, among which the sample transfected with F3'HP2 had the strongest GUS enzyme activity, followed by F3'HP1. In addition, the GUS enzyme activity of these two samples was significantly higher than that of the samples transfected with F3'HP3, F3'HP4 and F3'HP5. Figure 3 b It can also be seen that the GUS enzyme activity of the sample transfected with F3'HP3 is stronger than that of the samples transfected with F3'HP4 and F3'HP5, but the difference does not reach a significant level; the GUS enzyme activity of the samples transfected with F3'HP4 and F3'HP5 is equivalent to that of the control. The above results indicate that the segment between F3'HP1 and F3'HP2 may contain a motif that binds and inhibits the activity of F3'HP, resulting in the activity of F3'HP1 being lower than that of F3'HP2; the segment between F3'HP2 and F3'HP3 may be the key segment that affects the highest activity of F3'HP; in addition, only the promoter sequence of F3'HP5 cannot maintain the lowest activity of F3'HP.

[0043] Note: Capital letters indicate that the difference between two data reached an extremely significant level (P<0.01).

[0044] 1.4F3'H response to low temperature treatment

[0045] from Figure 1 and Figure 2 The promoter of F3'H gene was found to have a low temperature response element (LTR), a gibberellin response element (P-box) and multiple light response elements. When tea plants were treated at 10℃ for 6h (CA_6h), F3'H gene was down-regulated; when tea plants were cultured at 10℃ during the day and 4℃ at night for 7 days (CA_7d), F3'H gene was significantly up-regulated; when tea plants were cultured at 4℃ during the day and 0℃ at night for 7 days (FA_7d), F3'H gene was down-regulated, but the expression level was significantly higher than before cold acclimation (CK, 25℃ during the day, 20℃ at night, 12h photoperiod, relative humidity of 70%); after cold acclimation, the normal CK conditions were restored for 7 days, and the expression level of F3'H gene was significantly higher than CK. The above results indicate that F3'H gene is induced by low temperature, and the low temperature response element LTR on F3'HP may play an important role in it.

[0046] Note: CS: cold stimulation; CA: cold adaptation; FA: freezing acclimation; DA: acclimation.

[0047] 2 Discussions

[0048] F3'H is a key branch gene for catechin synthesis, catalyzing the formation of B-ring dihydroxy products from flavonoids. Therefore, the content of dihydroxycatechins in tea is closely related to the F3'H gene.

[0049] Promoters play a key role in the regulation of gene expression, and the activity level of promoters is affected by transcription factors that bind to various cis-acting elements in the promoter and the complexes they form (Vandel et al., 2019; Martinez-Corral et al., 2023). The present invention cloned the promoter sequence of 1806 bp upstream of the 'ATG' of the F3'H gene in the tea variety 'Yunkang 10', and found that in addition to the basic cis-acting elements including TATA-box and CAAT-box, the promoter sequence of the F3'H gene also has multiple and various other types of cis-acting elements ( Figure 1 and Figure 2 ). Transcriptome data showed that F3'H responded to low temperature, and cold acclimation upregulated the expression level of F3'H gene ( Figure 4 ), this result is consistent with the presence of a low temperature response element (LTR) on the F3'H promoter. The F3'H gene promoter of tea trees has multiple cis-acting elements related to light response, such as Box 4, Sp1, TCCC-motif and TCT-motif, indicating that the expression of the F3'H gene may be regulated by light. The results of the study by Gu Chenchen et al. (2017) showed that the expression level of the F3'H gene decreased significantly with the increase in the number of shading days; the study by Liu et al. (2018) also found that the expression level of the F3'H gene decreased significantly with the extension of shading time, and shading led to a decrease in the content of dihydroxycatechins. These results further indicate that the expression of the F3'H gene is regulated by light. In addition, the F3'H gene promoter sequence also has a gibberellin response element (P-box) and anaerobic induction-related elements (ARE and GC-motif) ( Figure 1 and Figure 2 ), which indicates that the F3'H gene may be induced by gibberellins and oxygen content.

[0050] Compared with the full-length promoter F3'HP1, the 5' fragment deleted F3'HP2 has stronger activity ( Figure 3), indicating that the segment between F3'HP1 and F3'HP2 may have a binding site for transcriptional repressors. Similar results were also found in studies on gene promoters such as Jatropha curcas GAST1 (Chen Yuqian, 2022), Vitis amurensis F3'5'H (Zhang Yu et al., 2022), and Jasmine TPS (Lin Junjie et al., 2022). The results of GUS histochemical staining and enzyme activity assay showed that after the segment between F3'HP2 and F3'HP3 was deleted, the activity of the F3'H promoter decreased sharply. The enzyme activity of F3'HP2 was 7.2 times that of F3'HP3, and the enzyme activity of F3'HP3 was not significantly different from that of the control, indicating that the segment between F3'HP2 and F3'HP3 is the key segment to ensure that the F3'H promoter has high activity. Although the enzyme activity of F3'HP3 is not significantly different from that of the control, it can be found from the results of staining and enzyme activity determination that F3'HP3 still has certain promoter activity, while F3'HP4 and F3'HP5 have no detectable activity, indicating that the segment between F3'HP3 and F3'HP4 may contain basic elements that meet the F3'H promoter activity. The present invention's exploration of the key segment of F3'H promoter activity provides a certain reference for analyzing the regulatory mechanism of the F3'H gene.

[0051] 3 Materials and methods

[0052] 3.1 Experimental Materials

[0053] The present invention uses the national fine variety 'Yunkang No. 10' (YK10) for cloning the F3'H gene promoter (F3'HP1), and the wild type Nicotiana benthamiana is preserved in the laboratory for genetic transformation and activity analysis of F3'HP-GUS. The cloning vector pMD19-T, the quick-cutting enzyme SaII, the quick-cutting enzyme Kan I, the competent Escherichia coli cell DH5α, and the competent cell EHA105 are all purchased from Baoriyi Biotechnology (Beijing) Co., Ltd., and the chemical reagents such as X-gluc are purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and the gene cloning uses the KOD-Plus-Neo enzyme of Toyobo (Shanghai) Biotechnology Co., Ltd. DNA extraction and gel recovery are all used with the relevant kits of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0054] 3.2 Cloning, 5' fragment deletion and bioinformatics analysis of the tea plant F3'H promoter

[0055] Primers were designed based on the 2000bp upstream coding region of the F3'H gene (CSS0048905) in the tea plant genome (Xia et al., 2020). The full-length promoter sequence of the F3'H gene was cloned from the genomic DNA of YK10 by PCR. Then, the 5' fragment of F3'HP1 was deleted by PCR. The deleted sequences were named F3'HP2, F3'HP3, F3'HP4 and F3'HP5 (the lengths were reduced in sequence). The obtained F3'HP1 sequence and the sequence after deletion were submitted to the Plant CARE website (Rombauts et al., 1999) to analyze the cis-acting elements of the promoter sequence; the gene sequence information was visualized using the software TBtools (Chen et al., 2020).

[0056] 3. Construction of GUS fusion expression vector containing 3'HP1 and its 5' end fragment deletion sequence and transient transformation of tobacco

[0057] In order to connect the F3'HP1 and its 5' fragment deletion sequence amplified in 1.2 to the plant binary transformation vector PBI101.3-GUS plus (kindly donated by Professor Chen Liang's research group of the Tea Research Institute of the Chinese Academy of Agricultural Sciences, denoted as PBI) to construct the GUS (β-glucuronidase gene) expression vector, SaII and Kan I restriction sites were added to the 5' end of the forward and reverse primers respectively. Then the target sequence was connected to the PBI vector by double restriction method. The method of tobacco transient transformation refers to the report of Li Yin et al. (2010). The primer sequences used are shown in Table 2.

[0058] Table 2 Primer sequences used in the experiment

[0059]

[0060] The lowercase letters in the sequence are protected bases; the underlined bases are restriction enzyme cleavage sites.

[0061] The lower case letters of the sequence are the protecting base; the underlined base is the restriction site.

[0062] 3.4 GUS histochemical staining and GUS enzyme activity assay of transiently transformed tobacco

[0063] Take the tobacco leaves after instantaneous transformation, add GUS reaction solution, keep warm at 37°C overnight (8-16h), add 70% alcohol for decolorization after staining, decolorize (2-3 times) until the negative control is colorless, and then observe and take pictures. The preparation method of GUS reaction solution refers to the report of Wang Zhen et al. (2021). The method of GUS enzyme activity determination refers to the report of Yan Qiang et al. (2022). Three tobacco plants were transformed with each gene, and three young leaves were transformed from each tobacco plant. The three leaves of each tobacco plant were used as a biological replicate. The GUS histochemical staining sampling was two tissues of about 0.3mm per leaf, and the rest were used as GUS enzyme activity determination samples.

[0064] Response of 3.5F3'H to low temperature treatment

[0065] The expression data of the F3'H gene of tea plants after low temperature treatment were derived from the transcriptome data (Li et al., 2019), and the gene expression level was expressed by TPM (Transcripts Per Kilobase of exonmodel per Million mapped reads).

[0066] like Figure 5 As shown, a method for analyzing the activity of tea plant F3'H promoter comprises:

[0067] Cloning and expression vector construction of S1, F3'HP1 and its 5' end fragment lacking promoter sequence;

[0068] S2, cis -acting element analysis of the promoter sequence of F3'HP1 and its 5' end fragment deletion;

[0069] S3, GUS histochemical staining and GUS enzyme activity assay of transiently transformed tobacco;

[0070] S4, Response of F3'H to low temperature treatment.

[0071] Example 1: Analysis of cis-acting elements of tea plant F3'H promoter

[0072] Through cis-acting element analysis, the potential regulatory mechanism of F3'H promoter in light response, low temperature response and gibberellin response was explored.

[0073] 1) Promoter sequence cloning

[0074] The F3'HP1 promoter sequence (1806 bp) was amplified by PCR from tea sample YK10, and specific primers were further designed to delete the 5' end of the fragment, obtaining fragments of 804 bp, 479 bp, 195 bp and 154 bp, which were named F3'HP2, F3'HP3, F3'HP4 and F3'HP5, respectively.

[0075] 2) Analysis of cis-acting elements

[0076] PlantCARE online analysis tool was used to predict the elements of F3'HP1 and its fragments. The results showed:

[0077] F3'HP1 contains multiple TATA-boxes, CAAT-boxes, light-responsive elements, low-temperature-responsive elements, and gibberellin-responsive elements.

[0078] As the fragments were deleted, the low temperature response elements and anaerobic induction related elements were deleted starting from F3'HP2.

[0079] F3'HP4 and F3'HP5 only retain a small amount of TATA-box, and the number of TATA-box in F3'HP5 is even smaller.

[0080] Through element analysis, it was found that the complete F3'HP1 promoter has abundant functional elements, while the deletion of the 5' end fragment resulted in a significant reduction in the types and number of elements. The reduction of elements may have a negative impact on the activity of the promoter, providing an important reference for subsequent functional verification.

[0081] Example 2: Analysis of the response of the F3'H promoter to low temperature

[0082] Verify the activity of F3'H promoter under low temperature stress conditions and its response characteristics to cold acclimation treatment.

[0083] #Experimental steps

[0084] 1) Low temperature treatment experiment

[0085] The tea tree samples were treated at 10℃ for 6 hours, cultured at 10℃ during the day and 4℃ at night for 7 days, and cultured at 4℃ during the day and 0℃ at night for 7 days.

[0086] After the treatment, RNA of the samples was extracted and transcriptome analysis was performed to determine the expression of the F3'H gene.

[0087] 2) Cold acclimation experiment

[0088] The tea plant samples were cold-acclimated at 4°C during the day and 0°C at night for 7 days and then returned to normal CK conditions for 7 days.

[0089] RNA was extracted and the expression changes of F3'H gene before and after cold acclimation were analyzed.

[0090] After treatment at 10°C for 6 hours, the expression of F3'H gene was down-regulated; however, after cultivation at 10°C during the day and 4°C at night for 7 days, the expression of F3'H gene was significantly up-regulated.

[0091] After further cultivation for 7 days under 4°C daytime and 0°C nighttime conditions, the expression level of F3'H gene was downregulated but remained higher than the level before cold acclimation.

[0092] After cold acclimation treatment, the expression level of F3'H gene was significantly higher than that of the control group when it returned to normal conditions, indicating that cold acclimation enhanced the promoter's ability to respond to low temperature.

[0093] This experiment verified the low temperature response ability of the F3'H promoter and the effect of cold acclimation treatment on its regulation, providing a basis for further research on the promoter activity of tea plants under low temperature stress.

[0094] Example 3: Analysis of F3'H promoter activity

[0095] Verify the activity differences of different segments of the F3'H promoter.

[0096] #Experimental steps

[0097] 1) Construction of GUS expression vector containing the full-length F3'H promoter and the 5' fragment deletion sequence

[0098] The target sequences were ligated to the PBI vector by double restriction digestion.

[0099] 2) Analysis of the activity of the full-length F3'H promoter and the 5' fragment deletion sequence

[0100] The vector constructed in 1) was used to transiently transform tobacco leaves. The GUS reaction solution was added to the tobacco leaves after transient transformation and kept at 37°C overnight (8-16h). After staining, 70% alcohol was added for decolorization (2-3 times) until the negative control was colorless, and then the leaves were observed and photographed.

[0101] The preparation method of GUS reaction solution refers to the report of Wang Zhen et al. (2021). The method of GUS enzyme activity determination refers to the report of Yan Qiang et al. (2022). Three tobacco plants were transformed with each gene, and three young leaves were transformed from each tobacco plant. The three leaves of each tobacco plant were used as a biological replicate. The GUS histochemical staining samples were taken from two tissues of about 0.3 mm per leaf, and the rest were used as samples for GUS enzyme activity determination.

[0102] This experiment verified that the F3'H promoter has strong activity, and the deletion of different promoter fragments has different effects on the activity of the F3'H promoter, which provides a basis for further research on the promoter activity of tea plants.

[0103] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for analyzing the activity of tea plant F3'H promoter, characterized in that: include: 1) Cloning and constructing expression vectors of F3'HP1 and its 5' end fragment with promoter sequence deleted; 2) cis-acting element analysis of the promoter sequence of F3'HP1 and its 5' end fragment deletion; 3) Chemical staining and GUS enzyme activity determination of GUS tissues of transiently transformed tobacco; 4) Analyze the response of F3'H gene promoter to low temperature treatment.

2. The method according to claim 1, wherein step 1 specifically comprises: Specific primers were designed by referring to the genomic sequence upstream of the start codon of the F3'H gene; The F3'HP1 promoter sequence and the 5' end fragment deletion sequence were cloned from the sample using PCR; The obtained sequence was inserted into an expression vector for subsequent experiments.

3. The method according to claim 1, wherein step 2 specifically comprises: The cis-acting elements of the promoter sequences of F3'HP1 and its deletion fragments were predicted using online analysis tools; Record and compare changes in the number and distribution of TATA-box, CAAT-box and other response elements in promoter sequences.

4. The method according to claim 1, wherein step 3 specifically comprises: Transiently transform tobacco leaves with the expression vector constructed with the promoter sequence; GUS histochemical staining was performed on transformed tobacco leaves; The staining intensity and GUS enzyme activity of samples transformed with different fragments were compared.

5. The method according to claim 1, wherein step 4 specifically comprises: Tea samples were treated with different temperature conditions, including short-term treatment at 10°C and cold acclimation cultivation with gradually decreasing temperatures; Transcriptome data were used to detect the expression level of the F3'H gene and analyze the promoter response under different temperature conditions.

6. The method according to claim 1, wherein: The fragment of F3'H promoter sequence includes the complete sequence F3'HP1, and the deleted sequences F3'HP2, F3'HP3, F3'HP4 and F3'HP5 with lengths of 804 bp, 479 bp, 195 bp and 154 bp, respectively.

7. The method according to claim 1, wherein: The expression vector of F3'H promoter was used to transiently transform tobacco samples to analyze the difference of promoter activity of different promoter fragments.

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